Therapeutic Efficacy of Interferon-Gamma and Hypoxia-Primed Mesenchymal Stromal Cells and Their Extracellular Vesicles: Underlying Mechanisms and Potentials in Clinical Translation.

Multipotent mesenchymal stromal cells (MSCs) hold promises for cell therapy and tissue engineering due to their self-renewal and differentiation abilities, along with immunomodulatory properties and trophic factor secretion. Extracellular vesicles (EVs) from MSCs offer similar therapeutic effects. However, MSCs are heterogeneous and lead to variable outcomes. In vitro priming enhances MSC performance, improving immunomodulation, angiogenesis, proliferation, and tissue regeneration. Various stimuli, such as cytokines, growth factors, and oxygen tension, can prime MSCs. Two classical priming methods, interferon-gamma (IFN-γ) and hypoxia, enhance MSC immunomodulation, although standardized protocols are lacking. This review discusses priming protocols, highlighting the most commonly used concentrations and durations, along with mechanisms and in vivo therapeutics effects of primed MSCs and their EVs. The feasibility of up-scaling their production was also discussed. The review concluded that priming with IFN-γ or hypoxia (alone or in combination with other factors) boosted the immunomodulation capability of MSCs and their EVs, primarily via the JAK/STAT and PI3K/AKT and Leptin/JAK/STAT and TGF-ß/Smad signalling pathways, respectively. Incorporating priming in MSC and EV production enables translation into cell-based or cell-free therapies for various disorders.

ECM-derived biomaterials for regulating tissue multicellularity and maturation.

Recent breakthroughs in developing human-relevant organotypic models led to the building of highly resemblant tissue constructs that hold immense potential for transplantation, drug screening, and disease modeling. Despite the progress in fine-tuning stem cell multilineage differentiation in highly controlled spatiotemporal conditions and hosting microenvironments, 3D models still experience naive and incomplete morphogenesis. In particular, existing systems and induction protocols fail to maintain stem cell long-term potency, induce high tissue-level multicellularity, or drive the maturity of stem cell-derived 3D models to levels seen in their in vivo counterparts. In this review, we highlight the use of extracellular matrix (ECM)-derived biomaterials in providing stem cell niche-mimicking microenvironment capable of preserving stem cell long-term potency and inducing spatial and region-specific differentiation. We also examine the maturation of different 3D models, including organoids, encapsulated in ECM biomaterials and provide looking-forward perspectives on employing ECM biomaterials in building more innovative, transplantable, and functional organs.

Efficacy and safety of small extracellular vesicle interventions in wound healing and skin regeneration: A systematic review and meta-analysis of animal studies.

Small extracellular vesicles (sEVs) have been proposed as a possible solution to the current lack of therapeutic interventions for endogenous skin regeneration. We conducted a systematic review of the available evidence to assess sEV therapeutic efficacy and safety in wound healing and skin regeneration in animal models. 68 studies were identified in Web of Science, Scopus, and PubMed that satisfied a set of prespecified inclusion criteria. We critically analyzed the quality of studies that satisfied our inclusion criteria, with an emphasis on methodology, reporting, and adherence to relevant guidelines (including MISEV2018 and ISCT criteria). Overall, our systematic review and meta-analysis indicated that sEV interventions promoted skin regeneration in diabetic and non-diabetic animal models and influenced various facets of the healing process regardless of cell source, production protocol and disease model. The EV source, isolation methods, dosing regimen, and wound size varied among the studies. Modification of sEVs was achieved mainly by manipulating source cells via preconditioning, nanoparticle loading, genetic manipulation, and biomaterial incorporation to enhance sEV therapeutic potential. Evaluation of potential adverse effects received only minimal attention, although none of the studies reported harmful events. Risk of bias as assessed by the SYRCLE's ROB tool was uncertain for most studies due to insufficient reporting, and adherence to guidelines was limited. In summary, sEV therapy has enormous potential for wound healing and skin regeneration. However, reproducibility and comprehensive evaluation of evidence are challenged by a general lack of transparency in reporting and adherence to guidelines. Methodological rigor, standardization, and risk analysis at all stages of research are needed to promote translation to clinical practice.

Scalable Production of Extracellular Vesicles and Its Therapeutic Values: A Review.

Extracellular vesicles (EVs) are minute vesicles with lipid bilayer membranes. EVs are secreted by cells for intercellular communication. Recently, EVs have received much attention, as they are rich in biological components such as nucleic acids, lipids, and proteins that play essential roles in tissue regeneration and disease modification. In addition, EVs can be developed as vaccines against cancer and infectious diseases, as the vesicle membrane has an abundance of antigenic determinants and virulent factors. EVs for therapeutic applications are typically collected from conditioned media of cultured cells. However, the number of EVs secreted by the cells is limited. Thus, it is critical to devise new strategies for the large-scale production of EVs. Here, we discussed the strategies utilized by researchers for the scalable production of EVs. Techniques such as bioreactors, mechanical stimulation, electrical stimulation, thermal stimulation, magnetic field stimulation, topographic clue, hypoxia, serum deprivation, pH modification, exposure to small molecules, exposure to nanoparticles, increasing the intracellular calcium concentration, and genetic modification have been used to improve the secretion of EVs by cultured cells. In addition, nitrogen cavitation, porous membrane extrusion, and sonication have been utilized to prepare EV-mimetic nanovesicles that share many characteristics with naturally secreted EVs. Apart from inducing EV production, these upscaling interventions have also been reported to modify the EVs' cargo and thus their functionality and therapeutic potential. In summary, it is imperative to identify a reliable upscaling technique that can produce large quantities of EVs consistently. Ideally, the produced EVs should also possess cargo with improved therapeutic potential.

Extracellular Vesicles in Facial Aesthetics: A Review.

Facial aesthetics involve the application of non-invasive or minimally invasive techniques to improve facial appearance. Currently, extracellular vesicles (EVs) are attracting much interest as nanocarriers in facial aesthetics due to their lipid bilayer membrane, nanosized dimensions, biological origin, intercellular communication ability, and capability to modulate the molecular activities of recipient cells that play important roles in skin rejuvenation. Therefore, EVs have been suggested to have therapeutic potential in improving skin conditions, and these highlighted the potential to develop EV-based cosmetic products. This review summarizes EVs' latest research, reporting applications in facial aesthetics, including scar removal, facial rejuvenation, anti-aging, and anti-pigmentation. This review also discussed the advanced delivery strategy of EVs, the therapeutic potential of plant EVs, and clinical studies using EVs to improve skin conditions. In summary, EV therapy reduces scarring, rejuvenates aging skin, and reduces pigmentation. These observations warrant the development of EV-based cosmetic products. However, more efforts are needed to establish a large-scale EV production platform that can consistently produce functional EVs and understand EVs' underlying mechanism of action to improve their efficacy.

Mesenchymal Stem Cell-Derived Exosomes and MicroRNAs in Cartilage Regeneration: Biogenesis, Efficacy, miRNA Enrichment and Delivery.

Exosomes are the small extracellular vesicles secreted by cells for intercellular communication. Exosomes are rich in therapeutic cargos such as microRNA (miRNA), long non-coding RNA (lncRNA), small interfering RNA (siRNA), DNA, protein, and lipids. Recently, many studies have focused on miRNAs as a promising therapeutic factor to support cartilage regeneration. Exosomes are known to contain a substantial amount of a variety of miRNAs. miRNAs regulate the post-transcriptional gene expression by base-pairing with the target messenger RNA (mRNA), leading to gene silencing. Several exosomal miRNAs have been found to play a role in cartilage regeneration by promoting chondrocyte proliferation and matrix secretion, reducing scar tissue formation, and subsiding inflammation. The exosomal miRNA cargo can be modulated using techniques such as cell transfection and priming as well as post-secretion modifications to upregulate specific miRNAs to enhance the therapeutic effect. Exosomes are delivered to the joints through direct injection or via encapsulation within a scaffold for sustained release. To date, exosome therapy for cartilage injuries has yet to be optimized as the ideal cell source for exosomes, and the dose and method of delivery have yet to be identified. More importantly, a deeper understanding of the role of exosomal miRNAs in cartilage repair is paramount for the development of more effective exosome therapy.

Biosafety evaluation of culture-expanded human chondrocytes with growth factor cocktail: a preclinical study.

The scarcity of chondrocytes is a major challenge for cartilage tissue engineering. Monolayer expansion is necessary to amplify the limited number of chondrocytes needed for clinical application. Growth factors are often added to improve monolayer culture conditions, promoting proliferation, and enhancing chondrogenesis. Limited knowledge on the biosafety of the cell products manipulated with growth factors in culture has driven this study to evaluate the impact of growth factor cocktail supplements in chondrocyte culture medium on chondrocyte genetic stability and tumorigenicity. The growth factors were basic fibroblast growth factor (b-FGF), transforming growth factor ß2 (TGF ß2), insulin-like growth factor 1 (IGF-1), insulin-transferrin-selenium (ITS), and platelet-derived growth factor (PD-GF). Nasal septal chondrocytes cultured in growth factor cocktail exhibited a significantly high proliferative capacity. Comet assay revealed no significant DNA damage. Flow cytometry showed chondrocytes were mostly at G0-G1 phase, exhibiting normal cell cycle profile with no aneuploidy. We observed a decreased tumour suppressor genes' expression (p53, p21, pRB) and no TP53 mutations or tumour formation after 6 months of implantation in nude mice. Our data suggest growth factor cocktail has a low risk of inducing genotoxic and tumorigenic effects on chondrocytes up to passage 6 with 16.6 population doublings. This preclinical tumorigenicity and genetic instability evaluation is crucial for further clinical works.

The Potential of Mesenchymal Stromal Cell as Therapy in Neonatal Diseases.

Mesenchymal stromal cells (MSCs) can be derived from various tissue sources, such as the bone marrow (BMSCs), adipose tissue (ADSCs), umbilical cord (UC-MSCs) and umbilical cord blood (UCB-MSCs). Clinical trials have been conducted to investigate the potential of MSCs in ameliorating neonatal diseases, including bronchopulmonary dysplasia (BPD), intraventricular hemorrhage (IVH) and necrotizing enterocolitis (NEC). In preclinical studies, MSC therapy has been tested for the treatment of various neonatal diseases affecting the heart, eye, gut, and brain as well as sepsis. Up to date, the number of clinical trials using MSCs to treat neonatal diseases is still limited. The data reported thus far positioned MSC therapy as safe with positive outcomes. However, most of these trials are still preliminary and generally smaller in scale. Larger trials with more appropriate controls and a longer follow-up period need to be conducted to prove the safety and efficacy of the therapy more conclusively. This review discusses the current application of MSCs in treating neonatal diseases, its mechanism of action and future direction of this novel therapy, including the potential of using MSC-derived extracellular vesicles instead of the cells to treat various clinical conditions in the newborn.